The invention relates to methods for screening for and identifying sequence alterations in nucleic acids.
The ability to scan and identify sequence alterations has widespread applications in many areas including genetics, immunology, infectious disease, oncology, epidemiology and forensics. Mutations leading to cancer can arise in a number of different genes and in different positions within the same gene. Gene variants also can be the source of hereditary diseases. Scanning and identification of such nucleic acid alterations have important implications for diagnosis and prognosis and for guiding therapy.
Several methods exist for scanning for the presence or absence of sequence variants. Denaturing Gradient Gel electrophoresis (DGGE) takes advantage of differences in the melting location of slightly different nucleotide sequences on a gradient gel during electrophoresis. (Mers et al., Methods Enzymol. 155:501-527 (1987); Abrams et al., Genomics 7:463-475 (1990); Vijg et al., U.S. Pat. No. 5,814,491) The process is generally performed on fragments of a nucleic acid of interest produced through polymerase chain reaction (PCR). Similarly, differences in mobility during electrophoresis of single strands of nucleic acid is used in the technique known as Single-Strand Conformation Polymorphism (SSCP). (Orita et al., Genomics 5:874-879 (1989)) This method takes advantage of different conformations assumed by nucleic acids having slightly different sequences under non-denaturing conditions. But, while these methods can identify nucleic acid sequences with alterations, the nature and exact location of the alteration(s) must be subsequently determined by other techniques.
Identification of the location and nature of an alteration in a nucleic acid sequence may be determined by direct sequencing. However, this process is generally very labor intensive and time-consuming. Other means of detecting known sequence alterations involve the use of oligonucleotides which hybridize to the specific altered sequence. (Conner et al., Proc. Nat. Acad. Sci., 80:278-282 (1983)) This method becomes untenable, however, when an alteration is unknown. Other methods utilizing restriction enzymes have been developed to identify alterations in a sequence without directly sequencing the nucleic acid. Unfortunately, these enzymes recognize only a limited number of restriction sequences. And none of these methods provides for quick determination of which alteration is actually present.
It is apparent that there is a need for a fast and simple method of screening nucleic acids for alterations and determining the precise location and nature of such an alteration.
Accordingly, it is an object of the present invention to provide novel methods of screening for sequence alterations in a target nucleic acid. The invention provides methods utilizing probes which hybridize to nucleic acid to detect alterations in a target sequence as compared to a control.
It is a further object of the invention to provide methods of screening for and determining the location of a sequence alteration in a target nucleic acid in a single step. These methods utilize probes which hybridize to nucleic acid to detect the existence and location of alterations in a target sequence.
An additional object of the invention is to provide methods of screening, determining the location and determining the nature of a sequence alteration in a target nucleic acid in a single step. These methods provide for the identification of specific base changes in the target sequence.
In accordance with the objectives outlined above, the present invention provides methods of screening and identifying alterations in a target nucleic acid sequence as compared with a control nucleic acid. Probes are produced which are complementary to and, therefore, hybridize to overlapping regions of a control nucleic acid. The methods are based on the fact that probes directed to a control sequence denature from a target sequence at a different temperature than from the control when the target sequence has an alteration (such as a point mutation) as compared with the control at the location at which the probe hybridizes.
In one aspect of the present invention, a method is provided for identifying a sequence alteration in a target nucleic acid as compared to a control nucleic acid. The method entails hybridizing a plurality of nucleic acid probes with said target nucleic acid, wherein said probes are complementary to different overlapping regions of said control nucleic acid. The melting temperature (Tm) of at least two overlapping probes is determined, as well as the difference between the melting temperature of each probe from the target nucleic acid and the control nucleic acid (xcex94Tm). The difference between the xcex94Tms of overlapping probes is determined as an indication of whether or not a sequence alteration exists in the target nucleic acid as compared to the control nucleic acid. In one embodiment, as few as two probes are required to examine a specific sequence alteration in target nucleic acid as compared with control nucleic acid.
In the method described above, the difference in xcex94Tm between at least two overlapping probes indicates the location of a nucleotide difference in the target nucleic acid as compared to the control nucleic acid. In addition, the difference in xcex94Tm between at least two overlapping probes indicates a substitution in the target nucleic acid sequence as compared to the control nucleic acid. Furthermore, the difference in xcex94Tm between at least two overlapping probes indicates the type of nucleotide substituted in the target nucleic acid sequence as compared to the control nucleic acid.
In another aspect of the invention, provided herein is a method for identifying a sequence alteration in a target nucleic acid as compared to a control nucleic acid. The method involves hybridizing a plurality of nucleic acid probes with the target nucleic acid; a first set of probes is complementary to regions of the control nucleic acid separated by one or more nucleotides and at least a second set of probes is complementary to regions of the control separated by one or more nucleotides. The regions complementary to the second set of probes include the nucleic acids separating the first set of probes and are overlapping with the regions complementary to the first set of probes. The method further entails determining the melting temperature (Tm) of at least two overlapping probes from the target nucleic acid, determining for these at least two overlapping probes the difference between the Tm from the target nucleic acid and the Tm from the control nucleic acid (xcex94Tm), and determining the difference in determined xcex94Tm between overlapping probes. The difference xcex94Tm between overlapping probes provides an indication of the presence or absence of a sequence alteration in the target nucleic acid as compared to the control nucleic acid. Preferably only two sets of probes are used.
Further to the method just described, the difference in xcex94Tm between at least two overlapping probes indicates the location of a nucleotide difference in the target nucleic acid as compared to the control nucleic acid. In addition, the difference in xcex94Tm between at least two overlapping probes indicates a substitution in the target nucleic acid sequence as compared to the control nucleic acid. Furthermore, the difference in xcex94Tm between at least two overlapping probes indicates the type of nucleotide substituted in the target nucleic acid sequence as compared to the control nucleic acid.
Also with respect to the method just described, in one embodiment only three probes are used, two from the first set which are complementary to adjacent regions and one from the second probe set which overlaps each of the probes from the first probe set. In this latest case, when the probes from the first probe set have a xcex94Tm of zero and the probe from the second probe set has a xcex94Tm other than zero, an alteration in the target is indicated as being in the nucleotides of the control nucleic acid sequence separating the regions to which the probes from the first probe set are complementary. In the case that one nucleotide separates adjacent regions to which the probes of the first probe set are complementary, a xcex94Tm of zero for two adjacent (non-overlapping) probes and a xcex94Tm other than zero for a probe which overlaps both adjacent probes indicated that the alteration is at the nucleotide separating the adjacent regions. In this last case, the xcex94Tm of the overlapping probe indicates both a substitution as the sequence alteration and the type of substitution.